Modular bioresorbable or biomedical, biologically active supramolecular materials

ABSTRACT

The present invention relates to a modular supramolecular bioresorbable or biomedical material comprising (i) a polymer comprising at least two 4H-units and (ii) a biologically active compound. Optionally, the supramolecular bioresorbable or biomedical material comprises a bioresorbable or biomedical polymer as third component to tune its properties (mechanical and bioresorption properties). The supramolecular bioresorbable or biomedical material is especially suitable for biomedical applications such as controlled release of drugs, materials for tissue-engineering, materials for the manufacture of a prosthesis or an implant, medical imaging technologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/527,690, filed Oct. 29, 2014, now Allowed, which is a Divisional ofU.S. patent application Ser. No. 11/913,470, now U.S. Pat. No.8,883,188, which is the U.S. National Phase of International PatentApplication No. PCT/NL2006/050107, filed May 3, 2006, published as WO2006/118461, which claims priority to European Patent Application No.05111018.7, filed Nov. 21, 2005, European Patent Application No.05103764.6, filed May 4, 2005, and U.S. Provisional Patent ApplicationNo. 60/679,671, filed May 11, 2005. The contents of these applicationsare herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to new supramolecular bioresorbable or biomedicalmaterials that are biologically active, as well as to a process toprepare such bioresorbable or biomedical materials in a supramolecularand/or modular way, in order to obtain materials that allow easyfine-tuning of the material properties, bioresorption properties and/orbioactivity by making use of reversible supramolecular interactions.More specifically, appearance, mechanical strength, elasticity,bioresorption and bioactivity are tuned by making use of thesereversible supramolecular interactions. The new materials of thisinvention can be used in a variety of biomedical applications that willbenefit from said properties including biomedical coating compositions.

BACKGROUND OF THE INVENTION

A wide variety of bioresorbable or biomedical materials are known thatare mostly based on aliphatic polyesters (Uhrich et al. Chem. Rev. 99,3181-3198, 1999). The mechanical properties of current bioresorbable orbiomedical materials are strongly related to their high molecularweights that are in general over 100 kDa, the presence of chemicalcross-links, and the presence of crystalline domains in these polymers.Although the crystalline domains are beneficial for the mechanicalproperties of the material (strength and elasticity), they do have astrong impact on the biodegradation process of the material as thebiodegradation of crystalline domains is in general very slow andcrystalline domains may cause immunological responses. Moreover, theneed for high molecular weight polymers, in order to get the desiredmaterial properties, usually implies that high processing temperaturesare required, and these are unfavorable as thermal degradation processesbecome more likely, especially when biologically active species areinvolved.

There are also several examples of biologically active species that havebeen covalently attached to polymers for biomedical uses. Especially,oligo-peptide based cell-adhesion promoters such as RGD-sequences havehad considerable attention in this respect. RGD-peptides have beencovalently attached to a synthetic polymer by copolymerizingRGD-containing monomers, in order to obtain biologically activepolynorbornenes (Grubbs et al., J. Am. Chem. Soc. 123, 1275, 2001).Unfortunately, in this way it was only possible to obtain biologicallyactive polynorbornenes, a polymer that is not bioresorbable, and oneneeds complex chemistry to change the specific biofunctionality. As aresult, one is limited in the amount and choice of (combinations) ofbiologically active molecules. Consequently, this approach lacks freedomin the choice of polymers and bioactivities.

The biologically active RGD-sequence has also been covalently attachedto alginates, a naturally occurring polysaccharide (Mooney et al.,Biomaterials 20, 45, 1999). The resulting hydrogel materials showenhanced proliferation of myoblast cells. However, specific carbodiimidechemistry is needed to introduce the bioactivity and only materialsbased on alginates can be used, thereby limiting the mechanical andbioresorbable or biomedical properties of the resulting material.Moreover, polymers from natural sources, such as polysaccharides, aregenerally costly and may show quality differences when different batchesare compared. As the production of synthetic polymers is morecontrolled, synthetic polymers are preferred because a constant qualitycan be ensured.

Further known in the art are biomedical coatings that are used toimprove the biocompatibility of medical devices. For example, stents maybe coated to reduce thrombosis (cf. for example U.S. Pat. No. 6,702,850,incorporated by reference) and implants may be coated to reduce therisks of rejection. Biomedical coatings may further comprisebiologically active agents that are released in a controlled manner.Such biomedical coatings may be prepared by mixing a biologically activeagent with a polymeric coating formulation.

A biological active agent that has been covalently attached to severalpolymers for biomedical coatings are heparin-derivatives. For exampleheparins have been copolymerized in polystyrene and poly(ethyleneglycol) systems (Feijen et al., J. Mater. Sci. Mat. Med. 4, 353, 1997),or heparins have been covalently attached to polyurethanes as disclosedin WO98/23307.

These heparin-polymer conjugates are used as anti-thrombogenic coatingsfor structures to be introduced into living systems. In both casesaromatic diisocyanates are used that are known for their toxicbiodegradation profile and a relative low amount of heparin is availableat the surface of the coating resulting in a low anti-thrombogenicactivity.

Although a strong anchoring of the biologically active molecules to thepolymer backbone is preferred in order to guarantee strong cell-adhesionor prolonged bioactivity, there are also materials in which biologicallyactive molecules are only mixed with polymers and are thus notcovalently attached to the polymer chain. As a consequence, thebiologically active molecules leak out of the material and, therefore,such materials only find uses in drug delivery applications. Examplesare hydrogels and microcapsules. Unfortunately, in hydrogels, the rateof drug delivery is hard to tune, while these systems generally sufferfrom poor material properties. Additionally, the chemical cross-links intheir structure limit their biodegradation behaviour. Microcapsules, onthe other hand, are prepared from polymers with high glass-transition ormelting temperatures, limiting their mechanical performance. Also,microcapsules frequently need bio-incompatible organic solvents toprocess them.

Another example of non-covalently attached biological active moleculesare heparins that are ionically bound to cationic coatings due toheparin's intrinsic negative charge caused by the presence ofcarboxylates and sulfonates in the molecule, as disclosed for example inU.S. Pat. No. 4,229,838. This method is however rather limited becausethe bio-active compound is leached over time from the surface due to therelative low ionic binding strength.

Alternatively, hydrophobic interactions have been used to non-covalentlyattach heparin to polymeric surfaces by end-group functionalizingheparin with an alkyl chain (Matsuda et al., Biomacromolecules, 2, 1169,2001). However, the hydrophobic interactions are rather poor, resultingin a fast decrease in activity due to leakage of the heparins from thepolymeric surfaces.

In general, “supramolecular chemistry” is understood to be the chemistryof non-covalent, oriented, multiple (at least two), co-operativeinteractions. For instance, a “supramolecular polymer” is an organiccompound that has polymeric properties—for example with respect to itsrheological behaviour—due to specific and strong secondary interactionsbetween the different molecules. These non-covalent supramolecularinteractions contribute substantially to the properties of the resultingmaterial.

Supramolecular polymers comprising (macro)molecules that bear hydrogenbonding units can have polymer properties in bulk and in solution,because of the H-bridges between the molecules. Sijbesma et al. (see WO98/14504 and Science 278, 1601, 1997) have shown that in cases where theself-complementary quadruple hydrogen unit (4H-unit) is used, thephysical interactions between the molecules become so strong thatpolymers with much better material properties can be prepared.

Several telechelic polymers have been modified with 4H-units before, ashas been published in Folmer, B. J. B. et al., Adv. Mater. 12, 874,2000, and in Hirschberg et al., Macromolecules 32, 2696, 1999. However,these polymers only contain the 4H-unit coupled at the ends of thepolymer chains. Consequently, the number of 4H-units in themacromolecule is limited by the amount of end groups to two, and thefunctional units are always located on the periphery of the polymer,limiting the mechanical properties of the resulting materials.

WO 02/034312 discloses polymers to which heparin is covalentlyattached.via functional groups.

WO 02/46260 discloses polyurethane based polymers with end capped4H-bonding units that are optionally grafted with additional 4H-bondingunits. The disclosed polymers can be used as hot melt adhesive orTPU-foam. WO 02/98377 discloses a cosmetic composition for care and/ortreatment and/or make-up of keratinous materials comprising in aphysiologically acceptable medium an efficient amount of a polymerhaving functional groups that are capable to bind to other functionalgroups by at least three hydrogen bridges. WO 02/98377 explicitly refersto WO 98/14504 and states that WO 98/14504 does not disclose a cosmeticuse of the polymers disclosed therein. WO 02/46260 and WO 02/98377 usecomparable or the same chemistry as described in Folmer et al. andHirschberg et al.

WO 2004/016598, incorporated by reference, discloses chemistry toacquire polymers with grafted quadruple H-bonding units. For example,polyacrylates and polymethacrylates with grafted 4H-units have beenproduced using different kinds of polymerization techniques. WO2004/016598 further discloses that these polymers are suitable forapplications related to personal care, surface coatings, imagingtechnologies, biomedical applications, e.g. materials for controlledrelease of drugs ad materials for tissue engineering and tabletformation, adhesive and sealing compositions, and thickening agents andbinders.

WO 2004/052963, incorporated by reference, discloses polysiloxanescomprising 4H-units in the polymer backbone. More precisely,polysiloxanes are disclosed having (a) 4H-units directly incorporated inthe polymer backbone, or (b) 4H-units pending from the polymer backbone,wherein the 4H-units are covalently attached via one linker through asilicon-carbon bond. However, the disclosed polymers are notbioresorbable.

Low molecular weight telechelic polycaprolactone endcapped with 4H-unitshas been described by Dankers et al. (Abstracts of Papers, 225th ACSNational Meeting, New Orleans, La., United States, Mar. 23-27, 2003; seePMSE, 88, 52, 2003). It was found that films of this material werebiocompatible based on the observed attachment of fibroblast cells tothe films. The study on the biodegradation of this polymer showed thepresence of crystallites which is not favourable for bioresorption.Moreover, in a paper by ten Cate et al. (Abstracts of Papers, 225th ACSNational Meeting, New Orleans, La., United States, Mar. 23-27, 2003; seePolymer Preprints, 2003, 44(1), 618) it was shown that the elasticity ofthe material was rather poor, as elongations beyond 130% were notpossible.

Hence there is a need for versatile supramolecular bioresorbable orbiomedical materials that have good and tunable mechanical propertiesand/or tunable biofunctionality. Additionally, it is desired that thesematerials are tunable in their biodegradation behavior. Furthermore, itis desired that they can easily be prepared and processed. The presentinvention addresses these needs by introducing a supramolecular modularapproach, wherein different ingredients (or modules or components) areblended—with each module contributing its own specific characteristic(i.e. mechanical performance, bioresorption, bioactivity, etc.)—toproduce a material displaying the combined characteristics. This modularapproach is usually not easily possible, but is enabled here, asquadruple hydrogen bonding units (4H-units) are used in at least one ofthe modules that are applied, resulting in contact between the modulesin the final material. The presented approach eliminates the need forextensive covalent synthesis, as blending experiments with the variousmodules can be used to fine-tune the properties of the final material.In addition, every module can be prepared in a controlled way, leadingto well defined structures that result in products of controllable highquality.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel supramolecularbioresorbable or biomedical materials as well as the process to preparesuch materials with the aim to obtain biomedical materials with bettercharacteristics than those of the prior art. In particular, thesupramolecular biomedical material is a supramolecular coatingcomposition.

It is another object of the present invention to provide supramolecularbioresorbable or biomedical materials having the additional feature thatthey are easily fine-tuned with respect to their characteristics (e.g.mechanical properties, bioresorption, bioactivity, etc.). The presentinvention therefore relates to a supramolecular bioresorbable orbiomedical material comprising the following components:

(a) a polymer comprising at least two 4H-units; and(b) a biologically active compound;wherein the 4H-unit is represented by the general formulas (1) or (2):

wherein the C—X_(i) and the C—Y_(i) linkages each represent a single ordouble bond, n is 4 or more, and X_(i) represent donors or acceptorsthat form hydrogen bridges with the H-bridge forming monomeric unitcontaining a corresponding general form (2) linked to them with X_(i)representing a donor and Y_(i) an acceptor and vice versa. The structureof these 4H-units is in detail disclosed in WO 98/14505 which isexpressly incorporated by reference.

Component (a) is preferably bioresorbable when the supra, olecualrmaterial is bioresorbable, and according to the present invention, theterms “bioresorbable” and “bioresorption” encompasses processes such ascell-mediated degradation, enzymatic degradation and/or hydrolyticdegradation of the supramolecular bioresorbable polymer, and/orelimination of the supramolecular bioresorbable polymer from livingtissue as will be appreciated by the person skilled in the art.

In addition, a biologically active compound is to be understood as acosmetically and/or pharmaceutically active compound that can induce abiological or biochemical effect in a mammal, but does not includebiological systems such as cells and cell organelles.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows cell adhesion and spreading in vitro. Fibroblast cell(5·104 cells/cm2) adhesion and cell spreading on different drop castfilms (a: example 29a, b: example 29b, c: example 29c and d: example 14)after two days of cell culturing in the absence of FBS. In all cases 4mole % of peptide was mixed with the polymer of example 14. The cellswere visualized on the polymer films with optical microscopy; scale barsrepresent 100 μm.

FIG. 2 shows in-vivo behaviour of the supramolecular bioactivematerials: Solution-cast supramolecular bioactive films of examples 36a(a) and 36c (b), and the bare polymers of examples 14 (c) and 8 (d) weresubcutaneously implanted in male AO rats. The films and surroundingtissue are shown after 21 days of implantation. The samples were stainedwith toluidine blue for histological examination. The magnifications are200 times. The material is indicated with m. The fibrous capsule isshown with c. Blood vessels are indicated with v. The giant cells thatare budding into the material from the interface are indicated with anasterisk (*).

DETAILED DESCRIPTION OF THE INVENTION

When investigating supramolecular polymers comprising quadruple hydrogenbonding units (4H-units), it was surprisingly found that by blendingdifferent polymers, optionally modified with 4H-units, not only themechanical properties of the blends could be modified and improved, butalso their biodegradation behaviour. Moreover, biologically activecompounds, optionally modified with 4H-unit(s), could be added to thesematerials, making the bioresorbable or biomedical material biologicallyor biochemically active by blending in the desired biologically activecompound. This invention therefore enables the use of biologically orbiochemically active materials with improved mechanical properties,while being able to tune separately the biodegradation rate and thebioactivity of the material. Thus, this invention surpasses the state ofthe art in biomedical materials, as a simplified way of designing andpreparing new biologically active, bioresorbable or biomedical materialsis introduced by using the supramolecular modular approach.

Component (a)

Accordingly, component (a), is a polymer comprising at least two4H-units, preferably 2-50, more preferably 3-50, even more preferably3-20, and most preferably 4-15 4H-units that are covalently attached tothe polymer chain. The 4H-units may be attached at the termini of thepolymer chain as well as to the backbone of the polymer chain or both.Obviously, the supramolecular bioresorbable or biomedical material ofthis invention may comprise more than one component (a), e.g. polymersof different chemical nature, of different molecular weight, and/ordifferent numbers of 4H-units. It is also possible that component (a) isconstituted from components of different chemical nature and/or ofdifferent molecular weight.

It is preferred that component (a) is a bioresorbable polymer. However,if the supramolecular biomedical material is a supramolecular biomedicalcoating composition, it may be more preferred that component (a) is notbioresorbable.

Component (a) can be any type of polymer, i.e. the polymer can be ofsynthetic origin or of natural origin, such as chitosan, collagen,fibrin, or proteoglycans. However, it is preferred that component (a) isselected from the group consisting of polyethers, aliphatic polyesters,aromatic polyesters, polyurethanes, polyamides, polyacrylates,polymethacrylates, polyacrylamides, (hydrogenated) polyolefins,polysiloxanes, polycarbonates, polyorthoesters, polysaccharides,poly(N-vinylcaprolactam), polyvinylpyrrolidone and polyvinylalcohols(preferably partly esterified) or copolymers from these polymers such aspolyvinylpyrrolidone/vinyl acetate copolymer.

According to a more preferred embodiment of the invention, component (a)is selected from the group consisting of polyethers, aliphaticpolyesters, polycarbonates, polysiloxanes and polyorthoesters. Even morepreferably, component (a) is selected from the group consisting ofpolyethers, aliphatic polyesters and polycarbonates. Most preferably,component (a) is an aliphatic polyester.

In another more preferred embodiment of this invention, component (a) isselected from the group consisting of polyamides, polyacrylates,polymethacrylates, polyacrylamides, poly(N-vinylcaprolactam), orcopolymers of these polymers.

The number average molecular weight M_(n) of component (a) is preferablyin the range from 100 to 100000, more preferably from 100 to 60000, evenmore preferably 800 to 40000, most preferably from 2000 to 35000 Dalton.

Preferably, component (a) is prepared from relatively low molecularweight polymers having two hydroxy end-groups, primary amino end-groups,or a combination thereof. More preferably, component (a) is preparedfrom relatively low molecular weight polymers having two hydroxylend-groups. Examples of relatively low molecular weight polymers havingtwo hydroxy end-groups are:

(i) polyether diols having a polyoxyalkylene structure and OHend-groups;(ii) polyesters and copolyesters having OH end-groups;(iii) polycarbonates and copolycarbonates having OH end-groups;(iv) polyorthoesters having OH end-groups;(v) (hydrogenated) polyolefine diols; and(vi) polymers and copolymers based on combinations of these preferredpolymers (i)-(v).

Suitable examples of polymers (i) are polyetherdiols having apolyoxyalkylene structure and OH end-groups, e.g. polyethylene glycol,polypropylene glycol, poly(ethylene-co-propylene) glycol (random orblock), polytetramethylene glycol. Examples of polymers (ii) arepolyesters and copolyesters made by polycondensation of dicarboxylicacids, e.g. adipic acid, and diols, e.g. 1,6-hexanediol or glycols, orby polycondensation of hydroxyacids, e.g. lactic acid; polyesters andcopolyesters made by ringopening polymerisation of e.g. ε-caprolactone,glycolide, lactide, δ-valerolactone, 1,4-dioxane-2-one,1,5-dioxepan-2-one, oxepan-2,7-dione, and the like. Examples of polymers(iii) are polycarbonates and copolycarbonates based on e.g.1,6-hexanediol polycarbonate, polycarbonates and copolycarbonates madeby ringopening polymerization of e.g. trimethylenecarbonate,1,3-dioxepane-2-one, 1,3-dioxanone-2-one,1,3,8,10-tetraoxacyclotetradecane-2,9-dione. An example of polymers (iv)is a polyorthoester based on e.g.3,9-diethylene-2,4,8,10-tetraoxaspiro[5.5]undecane. Examples of polymers(v) are OH functionalized polybutadiene and OH functionalizedpoly(ethylene-butylene). An example of polymers (vi) are OHfunctionalized block copolymers of polycaprolactone andpolyethyleneglycol.

Examples of relatively low molecular weight polymers having two aminoend-groups are Jeffamines® (polyoxyalkylene amines produced and marketedby Huntsman), or other polyethers, aliphatic polyamides orpolysiloxanes.

Preferably, the polymers have two hydroxyl end-groups, primary amineend-groups, or a combination thereof have a number average molecularweight M_(n) of 500 to and 10000, more preferably of 750 to 7000.

According to a first preferred embodiment of the present invention, thesupramolecular bioresorbable or biomedical material comprises 50.0-99.99percent by weight of component (a) and 0.01-50.0 percent by weight ofcomponent (b) if no component (c) is present (vide infra). Morepreferably, the supramolecular bioresorbable or biomedical materialcomprises 70.00-99.99 percent by weight of component (a) and 0.01-30.00percent by weight of component (b). Most preferably, the supramolecularbioresorbable or biomedical material comprises 90.00-99.95 percent byweight of component (a) and 0.05-10 percent by weight of component (b).All these weight ranges are based on the total weight of thesupramolecular bioresorbable or biomedical material.

Component (a) may have all kinds of different architectures, e.g. alinear (co)polymer with the 4H-units attached to it as end groups,and/or in the polymer backbone, and/or grafted onto the polymer chain; astar shaped (co)polymer with the 4H-units somehow covalently attached toit, preferably as end groups; a dendritic structure with the 4H-unitsattached to it as end groups, and/or in the dendritic arms; or a(multifunctional) branched or hyperbranched structure with the 4H-unitsattached to it as end groups, and/or in the branches. The (co)polymersmay have any kind of microstructure, such as a random, a block, asegmented or a randomly segmented structure, with the 4H-units attachedto this co-polymer in any fashion, such as end-capped, incorporated inthe polymer chain or grafted from the backbone.

In a preferred embodiment of this invention, component (a) comprises astar shaped polymer that is (partly) end-functionalized with 4H-units,or component (a) comprises a linear polymer to which several 4H-unitsare grafted, or component (a) comprises a linear (co)polymer with the4H-units attached to it as end groups and in the polymer backbone. Thepreferred ranges of the number of 4H-units are disclosed above.

More preferably, component (a) comprises a linear (co)polymer with the4H-units attached to it as end groups and in the polymer backbone. Mostpreferably, component (a) comprises a linear (co)polymer with the4H-units attached to it in the polymer backbone.

It is furthermore preferred to use components (a) with relative lownumber average molecular weights M_(n), preferably in the range from 100to 100000, more preferably from 100 to 60000, even more preferably 800to 40000, most preferably from 2000 to 35000, in order to allowmelt-processing of the supramolecular bioresorbable or biomedicalmaterial at temperatures preferably lower than 200° C., more preferablylower than 150° C., and most preferably lower than 100° C., or toprocess them from solutions at concentrations higher than 10% by weight,preferably higher than 15% by weight.

Optionally, ionic or ionogenic groups may be incorporated in component(a) in order to make the material more hydrophilic and therebyfacilitating water-solubility or water swelling of the supramolecularbioresorbable or biomedical material (i.e. gelling). Preferred ionogenicgroups are N-methyl-diethanolamine, 2,6-bis-(hydroxymethyl)-pyridine and2,2-bis(hydroxymethyl)-propionic acid.

In addition, component (a) may contain one or more hydrophilic polymericblocks in its polymer chain in order to facilitate water-solubility orwater swelling of the supramolecular bioresorbable or biomedicalmaterial (i.e. gelling). These hydrophilic polymeric blocks arepreferably derived from polyethylene glycol polymers, preferably havinga number average molecular weight M_(n) from 200 to 50000, morepreferably from 500 to 6000.

Component (a) can in particular be used to tune the mechanicalproperties of the supramolecular bioresorbable material of thisinvention. In a preferred embodiment of this invention, component (a)has an elongation at break of at least 140%. In another preferredembodiment of this invention component (a) has an E-modules >35 MPa.

Preferably, component (a) contains at least three 4H-units on average tocounterbalance components (b) if component (b) has less than two4H-units, the latter optionally acting as supramolecular chain stopper.

The 4H-unit

It is preferred that in formulas (1) and (2) n equals 4 so and that the4H-unit comprises four donors or acceptors in the arrays X₁ . . . X₄ andY₁ . . . Y₄. The 4H-unit may be self-complementary (i.e. the twohydrogen bonded units have an equal array of donors and acceptors), ornon self-complementary (i.e. the two hydrogen bonded units have twodifferent arrays of donors and acceptors). Preferably, the 4H-unitcomprises two successive donors, followed by two successive acceptors,i.e. it is preferred that X₁ and X₂ are donors and X₃ and X₄ areacceptors. Preferably, the donors and acceptors are O, S, and N atoms.The 4H unit is in detail disclosed in WO 98/14505 and in U.S. Pat. No.6,320,018 which are expressly incorporated by reference.

According to a preferred embodiment of the present invention the 4H-unithas the general formula (3) or formula (4), and tautomers thereof:

wherein X is a nitrogen atom or a carbon atom bearing a substituent R⁸,preferably a nitrogen atom, and wherein R¹, R², R³ and R⁸ areindependently selected from the group consisting of:(a) hydrogen;(b) C₁-C₂₀ alkyl;(c) C₆-C₁₂ aryl;(d) C₇-C₁₂ alkaryl;(e) C₇-C₁₂ alkylaryl;(f) polyester groups having the formula (5)

-   -   wherein R⁴ and Y are independently selected from the group        consisting of hydrogen and C₁-C₆ linear or branched alkyl, n is        1-6 and m is 10 to 100;        (g) C₁-C₁₀ alkyl groups substituted with 1-4 ureido groups        according to the formula (6)

R⁵—NH—C(O)—NH—  (6)

-   -   wherein R⁵ is selected from the group consisting of hydrogen and        C₁-C₆ linear or branched alkyl;        (h) polyether groups having the formula (7)

-   -   wherein R⁶, R⁷ and Y are independently selected from the group        consisting of hydrogen and C₁-C₆ linear or branched alkyl and o        is 10-100;        (i) oligopeptide groups consisting of sequences of 1 to 50,        preferably 1 to 10, amino acids; and    -   wherein the 4H-unit is bonded to a polymer backbone via R¹, R²        and/or R³ (so that R¹, R² or R³ represent a direct bond) with        the other R groups representing, independently a side chain        according to (a)-(h).

According to the invention, R¹, R², R³ and R⁸ are preferablyindependently selected from the group consisting of groups (a)-(h)disclosed above.

In a first preferred embodiment, the 4H-unit is bonded to a polymerbackbone via R¹ (so that R¹ constitutes a direct bond), while R² and R³are independently any one of the groups (a)-(i) defined above,preferably group (b), more preferably 2-ethylpentyl or methyl and mostpreferably methyl. Most preferably, the 4H-unit is bonded to the polymerbackbone via R¹, whereas R² is any one of the groups (a)-(h) definedabove, more preferably group (b), even more preferably 2-ethylpentyl ormethyl and most preferably methyl, and R³ is hydrogen.

In a second preferred embodiment, the 4H-unit is bonded to a polymerbackbone via R¹ and R² (so that R¹ and R² constitute direct bonds),while R³ is any one of the groups (a)-(i) defined above, preferablygroup (a) or (b), more preferably group (a).

In a third preferred embodiment, the 4H-unit is bonded to a polymerbackbone via R¹ and R³ (so that R¹ and R³ constitute a direct bond),while R² is any one of the groups (a)-(i) defined above, preferablygroup (b), more preferably 2-ethylpentyl or methyl and most preferablymethyl. This third preferred embodiment if more preferred than the firstand second preferred embodiments.

As will be apparent to the person skilled in the art, the groups (b)-(i)defined above may be linear, branched or cyclic where appropriate.

Component (b)

Additionally, the supramolecular bioresorbable or biomedical material ofthe present invention comprises a biologically active compound as acomponent (b). Preferably, the component (b) is selected from the groupconsisting of biologically active compounds with at least one 4H-unit upto a maximum of ten 4H-units, preferably one to four, and mostpreferably two to four 4H-units. These 4H-units are covalently attachedto the biologically active compound.

If no component (c) is present (vide infra), then the amount ofcomponent (b) is 0.01 to 50.00 percent by weight and the amount ofcomponent (a) is 50.00-99.99 percent by weight, based on the totalweight of the supramolecular bioresorbable or biomedical material, as isdisclosed above. According to this embodiment, it is preferred that theweight range of component (a) is 70.00-99.99 percent by weight, and evenmore preferably 90.00-99.95 percent by weight, whereas the preferredweight range for component (b) is 0.01-30 percent by weight, and evenmore preferably 0.05-10.00 percent by weight. All these weight rangesare based on the total weight of the supramolecular bioresorbable orbiomedical material. Moreover, component (b) may comprise one or moredifferent biologically active compounds.

The biologically active compound can be any compound that displaysbioactivity as disclosed above. A ‘biologically active compound’, asused herein, includes a compound that is biomedically relevant. Itfurther provides a therapeutic, diagnostic, cosmetic, medicinal orprophylactic effect, a compound that effects or participates in tissuegrowth, cell growth, cell differentiation, cell signalling, cell homing,protein absorption, i.e. a compound that may be able to invoke abiological action, or could play any other role in one or morebiological processes. Such compounds include, but are not limited to,antimicrobial agents (including antibacterial and anti-fungal agents),anti-viral agents, anti-tumor agents, anti-thrombogenic agents,anti-coagulant agents, lubricating agents, imaging agents, drugs,medicines, hormones, immunogenic agents, growth factors, cytokines,chemokines, (fluorescent) dyes, contrast agents, nucleic acids such asfor example single or double stranded DNA and single or double strandedRNA, lipids, lipopolysaccharides, (poly)saccharides, vitamins, andpeptides, polypeptides and proteins in general, biotinylated compoundsor other compound that target biologically relevant molecules.

A non-limiting, preferred and important group of species that can beused as component (b) according to the present invention is formed bypeptides, polysaccharides and proteins. Peptides include di-, tri- andtetrapeptides as well as oligopeptides and polypeptides as is known tothe person skilled in the art.

In a preferred embodiment, component (b) comprises a growth factor, ananti-microbial agent, a thrombin inhibitor, or an anti-thrombogenicagent. A growth factor is defined as a protein or peptide that has abeneficial effect on the growth, proliferation and/or differentiation ofliving cells. According to a more preferred embodiment of thisinvention, the supramolecular bioabsorbable material is advantageouslyused as a scaffold for tissue engineering, wherein the growth factor isnon-covalently bound to a polymer.

Examples of preferred growth factors are Bone Morphogenetic Proteins(BMP), epidermal growth factors, e.g. Epidermal Growth Factor (EGF),fibroblast growth factors, e.g. basic Fibroblast Growth Factor (bFGF),Nerve Growth Factor (NGF), Bone Derived Growth Factor (BDGF),transforming growth factors, e.g. Transforming Growth Factor-.beta.1(TGF-.beta.1), and human Growth Hormone (hGH).

If the supramolecular material is a supramolecular biomedical material,it is according to another preferred embodiment used as a biomedicalcoating composition, wherein the anti-thrombogenic agent isnon-covalently bound to a polymer. Non-limiting examples of preferredanti-thrombogenic agents are heparin, heparin analogues, heparincomplexes, and molecules comprising a sulfonated glycosaminoglycanmoiety. The anti-thrombogenic agent may also be a heparin covalentlybonded to one or more polymers as disclosed in WO 02/34312, incorporatedby reference herein. A preferred class of anti-thrombogenic agentsconsists of heparin, heparin analogues, heparin complexes, moleculescomprising a sulfonated glycosaminoglycan moiety, and heparinisedpolymers as disclosed in WO 02/34312.

Further examples of peptides or proteins which may advantageously beincluded in the supramolecular bioresorbable or biomedical materialinclude immunogenic peptides or immunogenic proteins, e.g. toxins, viralsurface antigens or parts of viruses, bacterial surface antigens orparts of bacteria, surface antigens of parasites causing disease orportions of parasites, immunoglobulins, anititoxins, antigens.

Although, in view of the thermal instability of peptides,polysaccharides and proteins, the method according to the presentinvention is particularly useful for preparing materials loaded withpeptides, polysaccharides and proteins, it is obviously also possible toload the supramolecular bioresorbable or biomedical material with asubstance other than a peptide, a polysaccharide or a protein. Suchbiologically active agents which may be incorporated includenon-peptide, non-polysaccharide and non-protein drugs and inorganiccompounds. It is possible within the scope of the present invention toincorporate drugs of a polymeric nature, but also to incorporate drugsor vitamins of a relatively small molecular weight of less than 1500, oreven less than 500.

Examples of non-peptide, non-polysaccharide or non-protein drugs whichmay be incorporated include the following: anti-tumor agents,antimicrobial agents such as antibiotics or hemotherapeutic agents,antifungal agents, antiviral agents, anti-inflammatory agents, anti-goutagents, centrally acting analgesics, local anesthetics, centrally activemuscle relaxants, hormones and hormone antagonistics, corticosteroidssuch as mineralocorticosteroids or glucocorticosteroids, androgents,estrogens, progestins.

Examples of inorganic compounds, which may be incorporated, include, butare not limited to reactive oxygen scavengers or bone-extracts likeapatite or hydroxyapatite.

Component (b) can be used as such, or can be chemically modified withone or more 4H-units. This chemical modification can be done by regularorganic synthesis procedures, e.g. as coupling methods using succinimideesters, sulfhydryl reactive agents, azides, (thio)isocyanates,carbiodiimides, aldehydes, or Cu(I)-catalyzed Huisgen [2+3] dipolarcycloadditions, or by regular solid state synthesis procedures which areknown to the person skilled in the art. Moreover, in case of peptidesand proteins, this chemical modification can be done using nativechemical ligation with a peptide or protein containing a C-terminalthio-ester and a 4H-unit with a N-terminal cysteine, native chemicalligation is known to people skilled in the art.

Optionally, the 4H-unit can be bonded to component (b) via a(bio)degradable linker that can be cleaved in vivo. In such a way thenative component (b) is gradually released from the material, forexample to induce an enhanced therapeutic effect. Non-limiting examplesof cleavable linkers are esters or oligopeptides that are cleaved byenzymatic activity, such as the cleavage of the peptide Gly-Phe-Leu-Glyby cysteineproteases.

Additionally, two or more different components (b) may be present in thesupramolecular bioresorbable or biomedical material. This is especiallybeneficial when the bioactivity is based on multivalent and/orsynergistic interactions. A non-limiting example of such interaction isthe cell adhesion advantageously mediated by a combination of RGD andPHSRN peptides.

Component (c)

The supramolecular bioresorbable or biomedical material according to thepresent invention preferably also comprises a third component (c), saidthird component (c) being a bioresorbable polymer.

Preferably, this bioresorbable polymer comprises one up to a maximum offifty 4H-units, preferably one to thirty, more preferably two to twenty,and most preferably four to twenty. These 4H-units are covalentlyattached to the polymer chain. The supramolecular bioresorbable orbiomedical material of this invention can obviously comprise differenttypes of components (c), wherein these components are for example ofdifferent chemical nature and/or of different molecular weight, and cancontain different numbers of 4H-units. It is obviously also possiblethat these polymers are constituted from elements of different chemicalnature and/or of different molecular weight.

Component (c) may be any bioresorbable polymer. However, it is preferredthat component (c) is selected from the group consisting of polyethers(preferably aliphatic), aliphatic polyesters, aromatic polyesters,polyamides (preferably aliphatic; for example polypeptides),polycarbonates (preferably aliphatic), polyorthoesters, polysaccharides,polyvinylalcohols (preferably partly esterified). It is even morepreferred that component (c) is selected from the group consisting ofaliphatic polyethers, aliphatic polyesters, aliphatic polyamides,aliphatic polycarbonates, aliphatic polyorthoesters, polysaccharides andpartially esterified polyvinylalcohols. In another embodiment of thisinvention, component (c) contains any combination of polymer types, forexample combinations of the preferred group of polymers disclosed above.According to a preferred embodiment of the invention, the polymerbackbone is selected from the group consisting of polysaccharides,polyether and copolyethers based on, for example, ethyleneoxide,propyleneoxide, or tetrahydrofuran; polyesters and copolyesters made bypolycondensation, based on, for example, adipic acid and diols such asglycols or hydroxyacids, such as lactic acid; polyesters andcopolyesters made by ringopening polymerisation, based on, for example,ε-caprolactone, glycolide, lactide, δ-valerolactone, 1,4-dioxane-2-one,1,5-dioxepan-2-one, oxepan-2,7-dione; polycarbonates andcopolycarbonates based on, for example, 1,6-hexanediol polycarbonate;polycarbonates and copolycarbonates made by ringopening polymerizationbased on, for example, trimethylenecarbonate, 1,3-dioxepane-2-one,1,3-dioxanone-2-one, 1,3,8,10-tetraoxacyclotetradecane-2,9-dione; orpolyorthoesters, based on, for example,3,9-diethylene-2,4,8,10-tetraoxaspiro[5.5]undecane; polymers andcopolymers based on combinations of these preferred polymers. Alsodifferent combinations of these preferred polymers can be present incomponent (c).

The number average molecular weight M_(n) of component (c) is preferablyin the range from 100 to 100000, more preferably from 100 to 60000, evenmore preferably 800 to 40000, most preferably from 2000 to 35000 Dalton.

Component (c) can have all kinds of different architectures: a linear(co)polymer with the 4H-units attached to it as endgroups, and/or in thepolymer backbone and/or grafted onto the polymer chain; a star shaped(co)polymer with the 4H-units somehow attached to it, preferably asendgroups; a dendritic structure with the 4H-units attached to it asendgroups, and/or in the dendritic arms; or a (multifunctional) branchedor hyperbranched structure with the 4H-units attached to it asendgroups, and/or in the branches, preferably only as endgroups. Theco-polymers can have any kind of microstructure, such as a random, ablock, a segmented or a randomly segmented structure, with the 4H-unitsattached to this co-polymer in any fashion, such as end-capped,incorporated in the polymer chain or grafted from the backbone.

Preferably, component (c) comprises a star shaped polymer that is(partly) end-functionalized with 4H-units, a linear polymer to whichseveral 4H-units are grafted, or a linear (co)polymer with the 4H-unitsattached to it as end groups and in the polymer backbone. Morepreferably, component (c) comprises a linear (co)polymer with the4H-units attached to it as end groups and in the polymer backbone. Mostpreferably, component (c) comprises a linear (co)polymer with the4H-units attached to it in the polymer backbone.

Like component (a), ionic or ionogenic groups may optionally beincorporated in component (c) in order to make the material morehydrophilic and thereby facilitating water-solubility or water swellingof the material (i.e. gelling). Preferred ionogenic groups are disclosedfor component (a). In addition, component (c) may contain one or morehydrophilic polymeric blocks in its polymer chain in order to facilitatewater-solubility or water swelling of the material (i.e. gelling). Thesehydrophilic polymeric blocks are preferably derived from polyethyleneglycol polymers, preferably having a number average molecular weightM_(n) from 200 to 50000, and more preferably from 500 to 6000.

Method of Preparing the Supramolecular Bioresorbable or BiomedicalMaterial

The present invention also provides a method of preparing thesupramolecular bioresorbable or biomedical material. This methodcomprises blending component (a), which predominantly attributes to themechanical strength of the supramolecular bioresorbable or biomedicalmaterial, and component (b) which predominantly attributes to thebiological activity of the supramolecular bioresorbable or biomedicalmaterial. According to a preferred embodiment of the present invention,this method comprises blending component (a), component (b) andcomponent (c), the latter predominantly modifying and/or attributing tothe bioresorption of the supramolecular bioresorbable or biomedicalmaterial. The blending of components (a), (b) and (c) results insupramolecular bioresorbable or biomedical materials with the desiredmaterial properties. In particular, if all components comprise 4H-units,they will all strongly contribute to strong physical interactionsbetween the different components in the blend. In particular, it istherefore preferred according to the present invention that all threecomponents (a)-(c) have at least one 4H-unit. The blending of allcomponents can be done by conventional processes, i.e. solutionprocessing or melt-processing, or a combination of both.

The concept of supramolecular blending of the different components alsoallows tuning the biodegradation behaviour of the supramolecularbioresorbable or biomedical materials, as this behaviour is determinedby the degradation behaviour of all the added components.

Preparation and Processing of the Supramolecular Bioresorbable orBiomedical Material

According to the supramolecular modular approach, the supramolecularbioresorbable or biomedical material can be obtained in three differentways: method (i) comprises blending the different components (a), (b)and optionally (c) with each other in conjunction with a mediumconsisting of one or more solvents, in which these components aredissolved or dispersed, preferably dissolved. This first method (i) ispreferably followed by processes for dissolved polymers known in theart.

A second method (ii) comprises blending the different components (a),(b) and optionally (c) with each other in the bulk at elevatedtemperatures, preferably 40° to 150° C. (vide infra). This second method(ii) is preferably followed by solventless processes for polymers knownin the art.

A third method (iii) comprises a combination of methods (i) and (ii).Hence, method (iii) comprises for example first blending component (b)with component (c) according to method (i), followed by blendingcomponent (a) and the blend of components (b) and (c) according tomethod (ii). Alternatively, method (iii) comprises first blendingcomponent (a) with component (b) according to method (i), followed byblending component (c) and the blend of components (a) and (b) accordingto method (ii). The other alternatives will be apparent to the personskilled in the art.

According to an especially preferred embodiment of the invention,methods (i) and (ii) comprises the in situ preparation of components (a)and/or (c).

Processing according to method (i) can be done from organic solvents oraqueous media, depending on the solubility of different components.Preferably, a solvent or mixture of solvents is used that is acceptablefor biomedical uses, such as water, acetone, methyl ethyl ketone, THF,DMSO, NMP, supercritical CO₂ or aliphatic alcohols, such as ethanol. Thesupramolecular bioresorbable or biomedical material is preferablyobtained by solvent casting, dip-coating, freeze-drying, precipitationcasting, spray coating, painting, roll-coating, foaming, solventspinning, wet spinning, electro-spinning, micro-contact printing, inkjet printing, particulate-leaching techniques, phase-separationtechniques or emulsion processes.

If the supramolecular material is a biomedical coating composition, thechoice of solvent(s) should be such that the desired viscosity of thesolution for the coating process is obtained, preferably polar solventsshould be used to reduce hydrogen bonding between the polymers.Moreover, the solvent has preferably a low boiling point in order tofacilitate removal from the solvent(s) after the coating process, andthe solvent (or solvent mixture) has preferably only limited toxicity.Therefore, drying of the supramolecular material is required after thecoating process and is preferably followed by extensive washings withwater or water containing a pH-buffer.

As will be known by persons skilled in the art, special care needs to betaken to clean the substrate surface when the supramolecular material isapplied as a coating to this substrate. For example, wettability of thesubstrate can be improved by liquid etching techniques, such as the useof chromic acid, aqueous sodium hydroxide and fuming sulfuric acid, orplasma etching techniques.

Processing according to method (ii) is done at temperatures sufficienthigh to allow processing of the components although temperatures shouldbe not too high to prevent degradation of the different components,especially component (b). Preferably, processing temperatures are inbetween 40° C. and 150° C., most preferably in between 50° C. and 120°C. The supramolecular bioresorbable or biomedical materials arepreferably obtained by extrusion, reactive-extrusion, micro-extrusion,fused deposition modeling, moulding, lamination, film-blowing, reactioninjection molding (RIM), spinning techniques, rapid prototyping or bythermal or photocuring of a coating.

The amount of component (a) in the supramolecular bioresorbable orbiomedical material is preferably 50.00-99.99 percent by weight if nocomponent (c) is present. According to this embodiment, component (a) ismore preferably present between 70.00-99.99 percent by weight, and mostpreferably between 90.00-90.95 percent by weight

The amount of component (b) in the supramolecular bioresorbable orbiomedical material is preferably 0.01-50.00 percent by weight if nocomponent (c) is present. According to this embodiment, component (b) ismore preferably present between 0.01-30.00 percent by weight, and mostpreferably between 0.05-10.00 percent by weight.

If component (c) is present in the supramolecular bioresorbable orbiomedical material according to the invention, the weight ratios ofcomponents (a)-(c) are preferably as follows: 20-59.99 percent by weightof (a), 0.01-40.00 percent by weight of (b), and 0.01-40.00 percent byweight of (c). More preferably, the weight ratios of components (a)-(c)are 40.00-69.99 percent by weight of (a), 0.01-30.00 percent by weightof (b), and 0.01-30.00 percent by weight of (c). All percentages byweight enlisted here for the supramolecular bioresorbable or biomedicalmaterial are based on the total weight of the supramolecularbioresorbable or biomedical material.

Highly porous structures can be obtained from the supramolecularbioresorbable materials of this invention by techniques known in theart, such as freeze-drying, particulate leaching, e.g. by using salts orsugars, and electro-spinning. Highly porous (interconnecting) structuresor non-woven fabrics are beneficial towards cell-attachment orproliferation, and allow the growth of tissue inside the scaffold. Thesestructures can, for example, be used as porous scaffolds used intissue-engineering, as prosthesis or implants.

Optionally, the supramolecular bioresorbable or biomedical material canbe used to prepare a hydrogel, i.e. a gel in which the liquid is water.Hydrogels can be obtained by persons skilled in the art by balancing theratio of hydrophilic and hydrophobic components in components (a) andoptionally (c) in the formulation. Hydrogel materials have a high watercontent, potentially mimicking different roles of the extracellularmatrices in tissue. Consequently, hydrogels find many uses in biomedicalapplications such as controlled drug delivery, delivery matrices or ascoatings.

According to this invention, additional ingredients other than (a), (b),or optionally (c), may be added to the material such as excipients knownin the art such as for example anti-oxidants and pH-buffers.

Applications

The supramolecular bioresorbable or biomedical materials according tothe invention are preferably suitable for applications related tobiomedical applications. In particular, the supramolecular bioresorbablematerials are not only useful for controlled release of drugs or medicalimaging technologies (for example MRI), but also for cosmeticapplications and in agricultural applications, such as in herbicides andpest control.

On the other hand, the biomedical materials are in particular suitableas materials for tissue-engineering, materials for the manufacture of aprosthesis or an implant. More preferably, the supramolecular biomedicalmaterials are useful for biomedical coatings with controlled release ofdrugs, biomedical coatings that have anti-thrombogenic or anti-microbialactivity, biomedical coatings that have enhanced lubrication. Thebiomedical coating can be applied on prothesis, implants, stents,catheters, or other medical devices that come in contact with livingtissue. According to another more preferred application, thesupramolecular biomedical material is useful as filling material forcosmetic and in reconstructive plastic surgery.

EXAMPLES

The following non-limiting examples further illustrate the preferredembodiments of the invention. When not specifically mentioned, chemicalsare obtained from Aldrich.

Example 1 Preparation of Upy1

1,6-Hexyldiisocyanate (650 g) and methylisocytosine (or2-amino-4-hydroxy-6-methyl-pyrimidine, 65.1 g) were suspended in a2-liter flask. The mixture was stirred overnight at 100° C. under anargon atmosphere. After cooling to room temperature, a litre of pentanewas added to the suspension, while stirring was continued. The productwas filtered, washed with several portions of pentane and dried invacuum. A white powder was obtained. ¹H NMR (400 MHz, CDCl₃): δ 13.1(1H), 11.8 (1H), 10.1 (1H), 5.8 (1H), 3.3 (4H), 2.1 (3H), 1.6 (4H), 1.4(4H). FT-IR (neat): ν (cm⁻¹) □2935, 2281, 1698, 1668, 1582, 1524, 1256.

Example 2 Preparation of Upy2

2-Amino-4-hydroxy-5-(2-hydroxy ethyl)-6-methyl-pyrimidine (12 gram) wassuspended in IPDI (150 mL) and was stirred overnight at 90° C. under anargon atmosphere. A clear solution developed. The solution was cooledand precipitated in hexane. The solid was filtered, stirred in anotherportion of hexane, and then the product was isolated by filtration,washing with hexane and drying of the residue. Yield: 98%. ¹H NMR (400MHz, CDCl₃): δ 13.1 (1H), 11.9 (1H), 10.2 (1H), 4.8-4.5 (1H), 4.2 (2H),4.0-3.2 (3H), 3.1-2.9 (3H), 2.7 (2H), 2.3 (3H), 1.9-1.6 (4H), 1.4-0.8(26H). FT-IR (neat): ν (cm⁻¹) □2954, 2254, 1690, 1664, 1637, 1590, 1532,1461, 1364, 1307, 1257, 1034, 791. MALDI-TOF-MS, [M⁺]=614, [M+Na⁺]=636.

Example 3 Preparation of Upy3

A mixture of methylisocytosine (10 g) and carbodiimidazole (20.7 g) indried DMSO (50 mL) was heated and stirred at 100° C. under an argonatmosphere for 2 hours. The resulting solid was filtered and washed withdry acetone until a white powder remained in the filter thatsubsequently was dried in vacuo and stored over P₂O₅. FT-IR (neat): ν(cm⁻¹) 3174, 1701, 1644, 1600, 1479, 1375, 1320, 1276.

Example 4 Preparation of Upy4

6-(2-Ethylpentyl) isocytosine (0.42 g) was dissolved in 5 mL chloroform.To this clear solution 1,1-carbonyldiimidazole (CDI) (0.71 g) was added.The reaction mixture was stirred for 3 hours at room temperature. Theentire mixture was extracted three times with brine. The water layerswere combined and extracted with chloroform. The combined chloroformlayers were dried with Na₂SO₄ and filtrated. The remaining organic layerwas dried under reduced pressure resulting in a light yellow powder in ayield of 66%. ¹H NMR (400 MHz, CDCl₃): δ 8.8 (1H), 7.6 (1H), 7.1 (1H),5.8 (1H), 2.5 (1H), 1.7 (4H), 1.3 (4H), 1.0 (3H), 0.9 (3H).

Example 5 Preparation of Upy5

UPy3 (0.9 g) and 1,6-diaminohexane (0.54 g; 1.1 eq.) were stirred for 72hours at room temperature in 15 mL of THF. The mixture was kept underargon. Ethanol (25 mL) was added, and the suspension was stirred forhalf an hour. The solid was filtered, washed with several 10 mL portionsof ethanol and dried. Resulting in 0.86 g of2(6-aminohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidinone. ¹H NMR(400 MHz, D₂O with a drop of CH₃COOH): δ=5.9 (1H), 3.2 (2H), 2.9 (2H),2.2 (3H), 1.7-1.2 (8H).

Example 6 Preparation of Upy6

Methylisocytosine (5.2 gram) was added to isophoronediisocyanate (IPDI,50 mL) and subsequently stirred at 90° C. under an argon atmosphere for3 days. The resulting clear solution was precipitated in heptane. Thewhite gom was collected, heated in 150 mL heptane, cooled on ice andfiltered. The same procedure was repeated once more with the whiteresidue, resulting in a white powder consisting of ureidopyrimidinonewith one IPDI unit. This product (10.22 g) was dissolved in chloroform(20 mL), and thereafter hydroxy ethyl acrylate (HEA, 3.6 mL) and 1 dropof dibutyl tin dilaurate (DBTDL) were added. The mixture was stirred atan oil bath temperature of 65° C. for 4 hours, and was then cooled andfiltered. The filtrate was concentrated and dropped into an excess ofdiethylether. The precipitate was collected by filtration, and waswashed with diethylether. Drying in vacuo gave a solid product. ¹H NMR(400 MHz, CDCl₃): δ 13.1 (1H), 11.7-12.0 (1H), 9.8-10.0 (1H), 6.4 (1H),6.2 (1H), 5.8 (2H), 5.2 (1H), 4.3 (4H), 4.1-3.6 (1H), 3.1-2.9 (2H), 2.1(3H), 2.0 (3H), 1.8-1.5 (2H), 1.4-0.8 (13H) 1.9 (3H), 1.7-1.2 (8H).

FT-IR (neat): ν 3212, 2954, 1697, 1660, 1572, 1520, 1242, 1165.

Example 7 Polymer I with 4H-Units

Telechelic hydroxy terminated PEO-6000 (10.20 g) was heated in vacuo ina 3-neck flask to 120° C. for 120 minutes and subsequently cooled downto 80° C. UPy2 (1.25 g) and two drops of dibutyltindilaurate dissolvedin toluene (40 mL) were added to the polymer melt and the solution wasstirred overnight under argon at 80° C. The reaction mixture was dilutedwith 40 mL THF and precipitated into diethylether. The material is white(semi-crystalline), elastic and tough. ¹H NMR (300 MHz, CDCl₃/CD₃OD): δ4.1, 3.6, 2.8, 2.2, 1.8-1.4, 1.2-0.8.

Example 8 Polymer II with 4H-Units

Telechelic hydroxy terminated polycaprolacton with a molecular weight of1250 D (25.9 g, dried in vacuo), UPy2 (10.9 g) and two drops ofdibutyltindilaurate were dissolved in dry ethylacetate (130 mL) andstirred overnight at an oil bath temperature of 70° C. The next day,ethylacetate (70 mL) and ethanol (50 mL) were added to the reactionmixture, which was subsequently precipitated into ethanol. The polymerwas isolated after drying of the precipitate, resulting in an elasticmaterial. ¹H NMR (300 MHz, CDCl₃): δ 13.1, 12.0, 10.1, 4.5-3.8, 3.0,2.6-2.2, 2.0-0.8. SEC (THF, PS-standards): M_(n)=8.8 kD, D=2.

Example 9 Polymer III with 4H-Units

Telechelic hydroxy terminated PEO-3000 (12.78 g) was heated in vacuo ina 3-neck flask to 120° C. for 30 minutes, followed by the addition of 5drops dibutyltindilaurate and UPy1 (2.51 g). This heterogeneous reactionmixture was subsequently stirred with a mechanical stirrer under anargon atmosphere and heated to 140° C. After 10 minutes stirring at 140°C. a clear homogeneous viscous liquid was obtained that after coolingwas isolated as a hard brittle white material. ¹H NMR (400 MHz, CDCl₃):δ 13.1, 11.9, 10.1, 5.8, 5.0, 4.2, 3.8-3.3, 3.2, 3.1, 2.1, 1.6-1.2.FT-IR (neat): ν (cm⁻¹) 2882, 1698, 1663, 1588, 1527, 1466, 1342, 1100,962, 841. SEC (THF, PS-standards): M_(n)=2.9 kD, D=1.2 Example 10:Polymer IV with 4H-units Bis(aminopropyl) endblocked polysiloxane DMSA21 with a viscosity of 100-120 cSt was obtained from Gelest. UPy3 (1.5g) was added to a solution of DMS A21 (14.7 g) in tetrahydrofuran (200mL). This mixture was subsequently heated to an oil bath temperature of80° C. and stirred at this temperature for 16 h under an argonatmosphere. Chloroform (200 mL) was added to the reaction mixture thatwas subsequently filtered over silica. The clear filtrate was washedtwice with saturated sodium chloride solution in water. The organicfraction was dried over Na₂SO₄, filtered and dried in vacuo to obtain anoff-white, clear, elastic material. Molecular mass (Mn) is 5.0 kg/mol;molecular weight distribution 1.8, determined by gel permeationchromatography (polystyrene standards). ¹H NMR (400 MHz, CDCl₃): δ 13.1,11.9, 10.2, 5.9, 3.3, 2.3, 1.6, 0.6, 0.4-−0.1. FT-IR (neat): ν (cm⁻¹)2961, 1698, 1659, 1587, 1527, 1258, 1010, 780. SEC (THF, PS-standards):M_(w)=8.1 kD.

Example 11 Polymer V with 4H-Units

Kraton L-2203, produced by Kraton Polymers, (average molecular weightM_(n)=3400, 10 g) was dissolved in dry toluene (100 mL). To this mixturewas added UPy1 (1.75 g) and 2 drops of dibutyltindilaurate, subsequentlythe turbid mixture was stirred at 80° C. under an argon atmosphere for12 h. A sample was taken and checked for complete reaction with ¹H NMR(disappearance multiplet at 3.6 ppm). The viscous reaction mixture wascooled to 70° C. while stirring and 0.3 mL of water was added. Thereaction mixture was stirred for an additional hour, followed byprecipitation in methanol (viscosity of reaction mixture can be loweredby the addition of more ethanol). The white gum was collected and driedin vacuo to obtain a slightly yellowish transparent rubber. Yield: 94%;¹H NMR (CDCl₃): δ 13.1, 11.9, 10.1, 5.8, 4.9, 4.6, 4.1, 3.8, 3.3, 3.2,2.2, 1.6-1.1, 0.8.

Example 12 Polymer VI with 4H-Units

Telechelic poly(2-methyl-1,3-propylene adipate) (average molecularweight M_(n)=2.0 kD, hydroxy end groups, 5.55 g) was stripped threetimes with toluene and dissolved in toluene (25 mL) together with UPy2(1.31 g) and few drops dibutyltindilaurate. The mixture was heated to80° C. and stirred for 16 hours under an argon atmosphere. Subsequentlyit was verified with FT-IR whether the isocyanate-functions haddisappeared, and the polymer was isolated by precipitation from achloroform/methanol solution into ether and drying of the solid. ¹H NMR(300 MHz, CDCl₃): δ 13.1, 12.0-11.8, 10.1-9.8, 5.0-4.6, 4.3-3.8,3.4-2.8, 2.5-2.0, 1.9-1.6, 1.4-0.8. SEC (THF, PS-standards): M_(n)=15.5kD, D=1.7.

Example 13 Polymer VII with 4H-Units

A mixture of telechelic hydroxy terminated poly(2-methyl-1,3-propyleneadipate) with an average molecular weight of 2.0 kD (2.39 g) andtelechelic hydroxy terminated polycaprolactone with an average molecularweight of 2.0 kD (2.39 g), was stripped three times with toluene anddissolved in chloroform (25 mL) together with monomer UPy2 (1.18 g) andfew drops dibutyltindilaurate. The mixture stirred overnight at 60° C.,followed by confirming the absence of isocyanate functions with FT-IRspectroscopy, UPy3 (0.35 g) was added and the solution diluted with 20mL chloroform and put to reflux for another night. Again, it wasverified with FT-IR whether the isocyanate-functions had disappeared,and the polymer was isolated by precipitation from a chloroform/ethanolsolution into hexane and drying of the solid. ¹H NMR (300 MHz, CDCl₃): δ13.2-12.8, 12.1-11.8, 10.2-9.8, 5.8, 5.2-4.5, 4.4-3.6, 3.4-2.6, 2.6-2.0,2.0-0.6. SEC (THF, PS-standards): M_(n)=12.2 kD, D=2.0.

Example 14 Polymer VIII with 4H-Units

Hydroxy-terminated polycaprolactone diol (M_(n)=2.1 kg/mole; obtainedvia ring-opening polymerization initiated by diethylene glycol;purchased from Acros) was dissolved in toluene, after which the toluenewas removed under reduced pressure to co-evaporate the water. Thisprocedure was repeated twice. This prepolymer (25.0 g; 12.5 mmol) wasdissolved in dry chloroform (750 mL) after which UPy1 (8.8 g) was added.After addition of two drops of dibutyltindilaurate the solution wasrefluxed for 16 hours. The completeness of the reaction was checked with¹H and ¹³C NMR for the presence of OH end-groups. Then 5 gram silicakieselgel 60 and two drops of dibutyltindilaurate were added and themixture was refluxed for 16 hours. With IR the absence of UPy1 in thesolution was checked. After dilution of the mixture with chloroform, thesilica was removed by filtration using hyflo. The solution wasconcentrated under reduced pressure. The material was precipitated fromchloroform (500 mL) in hexane (4.0 L) and filtrated. The resultingmaterial was dried for 24 hours in vacuo resulting in 24.4 g of 4H-unitcontaining telechelic polycaprolactone as a white fluffy material. ¹HNMR (CDCl₃): δ 13.1, 11.9, 10.1, 5.9, 4.9, 4.2, 4.1, 3.7, 3.2, 2.3, 2.2,1.6, 1.5, 1.4. FT-IR: ν=2941, 2865, 1729, 1699, 1669, 1587, 1527, 1461,1418, 1359, 1251, 1162, 1105 cm⁻¹

Example 15 Polymer IX with 4H-Units

Telechelic hydroxy terminated polycaprolactone with an average molecularweight of 1250 Dalton (10.94 g) was heated in vacuo in a 3-neck flask to120° C. for 30 minutes, followed by the addition of 8 dropsdibutyltindilaurate and UPy1 (5.13 g). This heterogeneous reactionmixture was subsequently stirred with a mechanical stirrer under anargon atmosphere and heated to 145° C. After 50 minutes stirring at 145°C. a homogeneous viscous paste was obtained that after cooling wasisolated as a hard white material. IR-spectroscopy confirmed that theproduct did not contained isocyanates anymore. ¹H NMR (400 MHz, CDCl₃):δ 13.2, 11.9, 10.2, 5.8, 4.9, 4.3, 4.1, 3.7, 3.4-3.1, 2.4-2.2, 3.1,1.8-1.2. FT-IR (neat): ν (cm⁻¹) 2882, 1698, 1663, 1588, 1527, 1466,1342, 1100, 962, 841. SEC (THF, PS-standards): M_(n)=2.1 kD, D=1.4

Example 16 Polymer X with 4H-Units

Telechelic hydroxy terminated polycaprolacton with a molecular weight of2.0 kD (9.73 g), UPy2 (2.5 g) and a few drops of dibutyltindilauratewere dissolved in chloroform (100 mL) and stirred overnight at an oilbath temperature of 60° C. The next day the chloroform was evaporated,toluene (100 mL) and pyridine (20 mL) were added, as well as a secondportion of UPy2 (0.5 g). The mixture was heated at an oil bathtemperature of 120° C. for another night, and the polymer product wasisolated by evaporation of the pyridine, precipitation fromchloroform/methanol 10:1 into methanol and drying of the solid. Uponstanding the material develops into a white (semi-crystalline), elasticpolymer. ¹H NMR (300 MHz, CDCl₃): δ 13.1, 12.0, 10.1, 4.5-3.8, 3.0,2.6-2.2, 2.0-0.8. SEC (THF, PS-standards): M_(n)=38.5 kD, D=2.0.

Example 17 Polymer XI with 4H-Units

Telechelic PEO-1500 (5.83 g) was stripped three times with toluene andwas then dissolved in toluene (30 mL). UPy2 (2.39 g) in toluene (14 mL)was added as well as a few drops of dibutyltindilaurate and the solutionwas heated overnight under argon (oil bath temperature of 120° C.). Thepolymer was isolated by precipitation into diethylether. The material iswhite (semi-crystalline), elastic and tough. ¹H NMR (300 MHz,CDCl₃/CD₃OD): δ 4.1, 3.6, 2.8, 2.2, 1.8-1.4, 1.2-0.8. SEC (THF,PS-standards): M_(w)=7.0 kD.

Examples of Bioactive Components (b) Example 18 UPy-GRGDS

A GRGDS peptide was synthesized according to conventional solid phasepeptide synthesis (SPPS) techniques using standard Fmoc-couplingchemistry on a Wang resin (the loading of the Wang resin withFmoc-Ser(tBu)-OH was 0.63 mmole/g; Bachem). In all cases, theFmoc-protection groups were deprotected with 20% piperidine in DMF. Theprotected (if necessary) amino acids (3 eq.; (Fmoc-Asp(OtBu)-OH,Fmoc-Gly-OH, Fmoc-Arg(Pmc)-OH and Fmoc-Gly-OH; Bachem) were dissolved inDMF. As coupling reagents 1-hydroxybenzotriazole (3.6 eq.) anddiisopropylcarbodiimide (3.3 eq.) in DMF were used. The coupling of the4H-unit to the free amine of the last amino acid (Gly) of the protectedGRGDS peptide was performed on the solid support using UPy3 (5 eq.) indry DMF (dried on molsieves) under an argon atmosphere for 16 hours at50° C. This resulted in the protected UPy-GRGDS on the resin. The excessof UPy3 was washed away with acidic water. The peptide was deprotectedand cleaved from the solid support with 95% trifluoro acetic acid (TFA)and 5% water. It was precipitated in (cold) diethylether, spun down andwashed three times with diethylether. Subsequently, the peptide wasfreeze-dried three times from water with 10-20% acetonitrile whichresulted in a white fluffy powder. UPy-GRGDS was purified usingpreparative reversed phase liquid chromatography (RPLC) if necessary.The compound was characterized with NMR techniques, IR, RPLC and massspectrometry. ¹H NMR (D₂O/ACN-d3): δ 5.98 (1H), 4.78 (1H), 4.48 (1H),4.32 (1H), 3.98-3.84 (2H), 3.15 (2H), 2.90-2.78 (2H), 2.23 (3H),1.85-1.61 (4H). The assignment of the ¹H NMR spectrum is confirmed by 2D¹H,¹H-COSY spectroscopy. ¹⁹F NMR (D₂O/ACN-d3), with potassium hexafluorophosphate as internal standard) showed that the sample contained lessthan 0.1 weight % TFA. FT-IR (neat): ν (cm⁻¹) 3280, 3182, 3073, 2948,2542, 1701, 1642, 1528, 1413, 1224, 1180, 1135, 1076, 1046. RPLC-MS: onepeak in chromatogram with m/z: Calcd. 641.3 g/mole. Obsd. [M+H]⁺=642.2g/mole and [M+H]²⁺=321.7 g/mole.

Example 19 Upy-Phsrn

The PHSRN peptide was synthesized according to conventional solid phasepeptide synthesis (SPPS) techniques using standard Fmoc-couplingchemistry on a Wang resin (the loading of the Wang resin withFmoc-Asn(Trt)-OH was 0.43 mmole/g; Bachem). In all cases, theFmoc-protection groups were deprotected with 20% piperidine in DMF. Theprotected (if necessary) amino acids (3 eq.; Fmoc-Arg(Pmc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-His(Trt)-OH and Fmoc-Pro-OH for PHSRN); Bachem)were dissolved in DMF. As coupling reagents 1-hydroxybenzotriazole (3.6eq.) and diisopropylcarbodiimide (3.3 eq.) in DMF were used. Thecoupling of the 4H-unit to the free amine of the last amino acid (Pro)of the protected PHSRN peptide was performed on the solid support usingUPy1 (8 eq.) in dry chloroform (molsieves) for 16 hours at 21° C. Thisresulted in the protected UPy-PHSRN on the resin. The excess of UPy1 waswashed away with acidic water. The peptide was deprotected and cleavedfrom the solid support with 95% trifluoro acetic acid (TFA) and 5%water. It was precipitated in (cold) diethylether, spun down and washedthree times with diethylether. Subsequently, the peptide wasfreeze-dried three times from water with 10-20% acetonitrile whichresulted in a white fluffy powder. UPy-PHRSN was purified usingpreparative reversed phase liquid chromatography (RPLC) if necessary. ¹HNMR (D₂O/ACN-d3): δ 8.58 (1H), 7.27 (1H), 5.92 (1H), 4.73 (1H), 4.66(1H), 4.36 (1H), 4.15 (1H), 3.86 (2H), 3.40-3.05 (2H), 2.82-2.73 (2H),2.21 (3H), 2.15 (1H), 2.03-2.00 (3H), 1.94-1.62 (4H), 1.54-1.46 (4H),1.34-1.27 (8H). The assignment of the ¹H NMR spectrum is confirmed by 2D¹H,¹H-COSY spectroscopy. ¹⁹F NMR (D₂O/ACN-d3), with potassium hexafluorophosphate as internal standard) showed that the sample contained lessthan 1 weight % TFA. FT-IR (neat): ν (cm⁻¹) 3263, 2943, 1657, 1542,1441, 1361, 1317, 1252, 1201, 1133, 1078. RPLC-MS: one peak inchromatogram with m/z: Calcd. 902.4 g/mole. Obsd. [M+H]⁺=903.3 g/mole,[M+H]²⁺=452.3 g/mole and [M+H]³⁺=301.9 g/mole.

Example 20 Upy-Fluorescein

UPy4 (0.51 g) was added to a solution of hexanediamine (2.03 g) inchloroform at room temperature. The mixture was stirred overnight. Basicwater (5 g NaOH in 20 mL water) was added to this mixture, and aftercentrifugation (5 min. at 4300 rpm) a clear water layer separated andwas subsequently isolated. The basic water layer was brought to pH=6with 3 M HCl in water. The amino-functional 4H-unit was isolated as awhite precipitate was formed, which was extracted with chloroform. Thechloroform layer was dried with Na₂SO₄ and evaporated. ¹H NMR (CDCl₃): δ13.25 (1H), 11.91 (1H), 10.21 (1H), 5.82 (1H), 3.27 (2H), 2.66 (2H),2.31 (1H), 1.69-1.28 (16H), 0.90 (6H). FT-IR (neat): ν (cm⁻¹) 3064;2956; 2856; 2927; 2856; 1939; 1664; 1594; 1558; 1520; 1428; 1265; 1178;1117; 1073; 950; 840; 814; 773; 756; 728; 697. Elemental analysis:C61.34, H9.64; N19.82, calculated (C61.51, H9.46, N19.92). RPLC-MS:[M+H]+=352.2 (calculated: 351.41); [isocytosine+H]⁺=210.2 (calculated:209.29); [UPy−C₆-UPy+H]⁺=587.3 (calculated: 586.78) g/mole.

This amino-functional 4H-unit (123 mg) was added tofluorescein-isothiocyanate (132 mg) in a 2:1 mixture of methanol andchloroform and stirred at room temperature for 2 days. The solvents wereremoved under reduced pressure. The remaining orange precipitate wasdissolved in 5 mL 0.2 M NaOH solution. Upon addition of 1.5 mL 1 M HClsolution a cloudy orange precipitate was formed. This was isolated bycentrifugation. Subsequently, the yellow clear water layer was pouredoff. The product was purified using a Sephadex LH 20 column (1:1dichloromethane: methanol). The product was dissolved in THF:water (1:1)and flushed over a small silica column. It was subsequentlyfreeze-dried. ¹H NMR (DMSO): δ 8.19 (1H), 7.91 (1H), 6.89 (1H), 6.81(1H), 6.58 (4H), 6.46 (2H), 5.72 (1H), 3.51 (2H), 3.18 (2H), 2.18 (1H),1.57-1.10 (16H), 0.74 (6H). LC-MS (direct injection): [M+H]⁺=741.2(calculated: 740.30); [2M+H]⁺=1481.1; [M_(fragment)+H]⁺=210.2;[M_(fragment)+H]⁺=352.3; [M_(fragment)+H]⁺=390.3;[M_(fragment)+H]⁺=707.3 g/mole.

Example 21 Upy-Biotin

To a solution of N-(+)-biotinyl-3-aminopropylammonium trifluoroacetate(165 mg, obtained from Sigma-Aldrich) in DMF (1.0 mL), Upy4 (124 mg) anddiisopropyl-ethylamine (DIPEA; 133 mg) were added. The mixture wasstirred at 60° C. for 7 hours. DMF was removed under reduced pressureand everything was dissolved in chloroform. The following extractionswere performed; 3 times with brine and 3 times with 1 M HCl. Thecombined organic layers were dried with Na₂SO₄ and concentrated to 1.5mL. This was precipitated in cold acetone and centrifugated. The acetonewas poured off and the product was dried under reduced pressure at 40°C. for 2 hours. This resulted in a white powder in a yield of 65%. ¹HNMR (400 MHz, CDCl₃): δ 13.32 (1H), 11.82 (1H), 10.07 (1H), 5.92 (1H),4.51 (1H), 4.36 (1H), 3.37 (4H), 3.13 (1H), 2.90 (1H), 2.73 (1H), 2.39(1H), 2.23 (2H), 1.81-1.19 (16H), 0.89 (6H). FT-IR (neat): ν (cm⁻¹)3217; 3038; 2929; 2860; 1699; 1641; 1583; 1526; 1460; 1439; 1381; 1306;1251; 1145; 1075; 1009; 951; 850; 796; 763; 739; 686. MALDI-TOF: 536.14(calculated 535.71 g/mole), 301.07, 334.23 g/mole.

Example 22 Upy-Gd(III)-Dtpa Complex

To a colourless solution of UPy4 (0.376 g, 1.24 mmol) in dichloromethane(5 mL) was slowly added a solution oft-butyl-6-amino-2-{{bis{2-[bis-(t-butoxycarbonylmethyl)amino]-ethyl}-amino}}hexanoate(0.713 g, compound obtained as described by: Anelli, P. L. et al.Bioconjugate Chem. 1999, 10, p. 137, compound 7). The solution wasvigorously stirred for 12 h at 20° C. The yellowish solution was washedwith 1 M KHSO₃ (aq) pH 1.95 (2×10 mL). Subsequently, the organic layerwas washed with 1 M K₂CO₃ (aq) pH 10 (3×10 mL) and brine (3×10 mL). Thecombined water layer was extracted with DCM (2×10 mL) and the organiclayer was dried over MgSO₄. The reaction mixture was concentrated underreduced pressure, yielding a yellowish liquid (0.84 g). The crudeproduct (0.730 g) was purified by column chromatography using EtOAc,yielding the protected SupraB-containing DTPA analogue in 0.51 g(R_(f)=0.5 (EtOAc)). ¹H-NMR (CDCl₃): δ 13.3 (1H), 11.9 (1H), 10.2 (1H),5.8 (1H), 3.5 (8H), 3.3-3.2 (3H), 3.0-2.6 (8H), 2.3 (1H), 2.0-1.2 (14H),0.94 (6H). The assignment of the 1H-NMR spectrum was confirmed by1H,1H-COSY. FT-IR (neat): ν (cm⁻¹) 2975, 2932, 1724, 1698, 1658, 1646,1586, 1526, 1367, 1253, 1219, 1148. ESI-QTOF-MS: m/z [C₅₀H₈₉N₇O₁₂+H]⁺Calcd. 980.67 Da, Obsd. 980.71 Da; [C₅₀H₈₉N₇O₁₂+Na]⁺ Calcd. 1002.65 Da,Obsd. 1002.65 Da.

This protected UPy-containing DTPA analogue was subsequently dissolvedin dichloromethane (0.39 g) and TFA (2 mL) was added, followed bystirring the reaction mixture for 16 h at room temperature. Afterevaporation of the solvent a second portion of TFA (2 mL) and drydichloromethane (5 mL) was added and stirring was continued overnight.The solution was concentrated in vacuo resulting in the TFA salt of thedeprotected product which was additionally purified by dialysis at 60°C. (100 Da MWCO membrane) followed by freeze-drying, yielding thepenta-acid as a white hygroscopic powder (0.266 g). ¹H-NMR (D₂O, 348 K):δ 6.2 (1H), 4.1 (8H), 3.6 (1H), 3.4 (4H), 3.3-3.1 (6H), 2.6 (1H),2.0-1.2 (14H), 0.79 (6H). The assignment of the 1H-NMR spectrum wasconfirmed by 1H,1H-COSY. ¹⁹F-NMR spectroscopy confirmed the successfulremoval of TFA by the absence of a signal at −75.6 ppm. FT-IR (neat): ν(cm⁻¹) 3215, 2933, 2531, 1700, 1630, 1551, 1431, 1389, 1333, 1202.ESI-QTOF-MS: m/z [C₃₀H₄₉N₇O₁₂+H]⁺ Calcd. 700.35 Da, Obsd. 700.40 Da;[C₃₀H₄₉N₇O₁₂+Na]⁺ Calcd. 722.33 Da, Obsd. 722.40 Da.

The desired Gd(III) complex was prepared by adding a stoichiometricamount of GdCl3·6 H2O (4.97 mg) in demineralised water (3 mL) to asolution of the penta-acid (9.16 mg) in 0.3 m citrate buffer at pH 5.8.The buffered solution was vigorously stirred for 2 h at roomtemperature. The aqueous solution was extensively dialysed (100 Da MWCOmembrane) and lyophilized. The resulting Gd(III) complex was obtained asa white hygroscopic powder (10.7 mg). FT-IR (neat): ν (cm⁻¹) 3384, 2932,1696, 1579, 1407, 1183, 1135, 1083. ESI-MS: m/z [C30H46N7O12Gd+H]+Calcd.855.25 Da, Obsd. 855.27 Da; [C30H45N7O12NaGd+H]+Calcd. 877.23 Da, Obsd.877.27 Da. [C30H44N7O12Na2Gd+H]+Calcd. 899.22 Da, Obsd. 899.27 Da.ICP-AES (Gd(III): Calcd. 50.0 μm, Obsd. 33.9 μm.

Example 23 Upy-Cysteine

A CGGKG peptide was synthesized according to conventional solid phasepeptide synthesis (SPPS) techniques using standard Fmoc-couplingchemistry on a Wang resin (the loading of the Wang resin withFmoc-Gly-OH was 0.75 mmole/g; Bachem). In all cases, the Fmoc-protectiongroups were deprotected with 20% piperidine in DMF. The protected (ifnecessary) amino acids (3 eq.; (Fmoc-Lys(Mtt)-OH, Fmoc-Gly-OH,Fmoc-Gly-OH and Fmoc-Cys(Trt)-OH; Bachem) were dissolved in DMF. Ascoupling reagents 1-hydroxybenzotriazole (3.6 eq.) anddiisopropylcarbodiimide (3.3 eq.) in DMF were used.

The 4H-unit was coupled to the Lys on the resin when the Cys was stillprotected. The Lys was first selectively deprotected in a mixture of 90%DCM/5% TFA/5% triisopropylsilane for 15 minutes. The resin was washedfor 2 times with DCM and for 4 times with DCM supplemented with 5%DIPEA. The coupling of the 4H-unit to the resulting free amine at theLys was performed on the solid support using UPy1 (8 eq.) in drychloroform (dried on molsieves) at a shaking table at 21° C. Thisresulted in the protected CGGK(UPy)G peptide on the resin. The excess ofUPy1 was washed away with acidic water. The Fmoc-group on the Cys wasremoved with 20% piperidine in DMF. The peptide was deprotected andcleaved from the solid support with 95% trifluoro acetic acid (TFA),2.5% water and 2.5% Tis. It was precipitated in (cold) diethylether,spun down and washed three times with diethylether. Subsequently, thepeptide was freeze-dried three times from water with 10-20% acetonitrilewhich resulted in a white fluffy powder. The compound was characterizedwith ¹H NMR and mass spectrometry. RPLC-MS: one peak in chromatogramwith m/z: Calcd. 713.8 g/mole. Obsd. [M+H]⁺=714.3 g/mole and an impurityof [M+H]⁺=820.3 g/mole and [M+H]²⁺=410.8 g/mole. ¹H NMR (400 MHz,D₂O/ACN-d3): δ 6.34 (1H), 4.79 (1H), 4.74 (1H), 4.41 (2H), 4.32 (4H),3.60 (2H), 3.44 (6H), 2.62 (3H), 2.31-2.04 (2H), 1.92-1.84-1.72 (12H).

Example 24 Upy-Heparine

Heparin sodium salt (1.0 g, Mn=12000, activity=195 IU/mg, PorcineIntestinal Mucosa, obtained from Merck Biosciences, Germany) wasdissolved in water and passed through a Dowex 50×8 (H+) column, followedby dialyzing (MW cut-off=12000-14000) against water and lyophilizationto obtain heparin (0.95 g). The carboxylic acid groups of heparin wereactivated by adding N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC)and N-hydroxysuccinimide (NHS) to 2% solution by weight of lyophilizedheparin in 0.05 M buffer of 2-morpholinoethane sulfonic acid(MES-buffer, pH=5.60), at a molar ratio of NHS:EDC:heparin-CO₂H of0.24:0.40:1.0. After 10 minutes pre-activation, UPy5 (64 mg) wasdissolved in MES-buffer (3 mL, pH=5.60) and added to the NHS/EDCactivated heparin solution (25 mL), resulting in a molar ratio of 6 to 1(UPy5 to heparin). After 3 h the reaction mixture was dialyzed onceagainst MES-buffer (pH=5.60), followed by extensive dialysis againstwater, followed by lyophilization to obtain heparin functionalized withapproximately six 4H-units.

Example 25 Upy-Heparine

Heparin sodium salt (1.0 g, Mn=12000, activity=195 IU/mg, PorcineIntestinal Mucosa, obtained from Merck Biosciences, Germany) wasdissolved in water and passed through a Dowex 50×8 (H+) column, followedby dialyzing (MW cut-off=12000-14000) against water and lyophilizationto obtain heparin (0.95 g). The reducing end of heparin was oxidizedwith iodide (0.2 g) in 20% aqueous methanol solution (25 mL) for 6 h atroom temperature. The reaction solution was added to ethanol containing4% by weight potassium hydroxide (50 mL). The resulting whiteprecipitate was filtered, dissolved in water and dialyzed (MWcut-off=12000-14000). Oxidized heparin was obtained after lyophilizationThe oxidized heparin was subsequently dissolved in water and passedthrough a Dowex 50×8 (H+) column followed by freeze drying to obtain thelactone-heparin (0.74 g). A 10-fold molar excess of UPy5 (45 mg) wasdissolved in DMF (2 mL) and subsequently added to lactone-heparin (200mg) dissolved in DMF (10 mL). The reaction was stirred for 16 h at 80°C. The reaction mixture was concentrated in vacuo followed by dissolvingin water. The dilute reaction mixture was subsequently passed through aDowex 50×8 (H+) column. The eluate was extensively dialyzed againstwater, followed by lyophilization resulting in heparin terminallyfunctionalized with a 4H-unit.

Examples of Processing of the Bioresorbable Supramolecular MaterialExample 26

The polymer of example 14 was processed via different techniques intoseveral scaffolds that can be issued for tissue-engineering. Films weremade by solvent casting from THF solution or via compression moulding(at approximately 20° C. above the melting temperature). Melt spinning(at a temperature of 90° C.) and electrospinning (from chloroformsolution) were used to make fibres and meshes. Grids with a fibre widthdown to approximately 220 m were produced via Fused Deposition Modelling(FDM) at temperatures just below 75° C.

Example 27

A bioactive hydrogel was obtained by dissolving the polymers of example7 (4.3 g) and of example 14 (1.0 g) in THF (70 mL), followed by thegentle addition of 82 mL deionized water to the stirredpolymer-solution. To this mixture was added Rhodamine B (100 mg). Thismixture was concentrated at a rotavap until all THF was removed and apink hazy hydrogel was obtained that showed orange fluoresence. Theresulting hydrogel had elastic properties and displayed viscoelasticbehavior.

Example 28

Bioactive materials were obtained by making three different peptidesolutions by dissolving: (a) 4 mole % of the oligopeptide of example 18;(b) 4 mole % of the oligopeptide of example 19; and (c) botholigopeptides of examples 18 and 19 together (4 mole % of each peptide)in THF with 10-30% water. Subsequently, the polymer of example 8 wasdissolved in THF. Bioactive blends were produced by mixing the peptidesolutions and polymer solution. The resulting mixtures were drop cast onglass cover slips (diameter=1.5 cm; 1·10⁻⁴ mmol peptide in the case of 4mole % peptide and 2.4·10⁻³ mmol polymer per cover slip) resulting inthree different oligopeptide-loaded films: 28a, 28b, and 28c. Most ofthe times, a slight precipitate was visible. The blends on the glasscover slips were dried in vacuo for 2-3 days at 35-40° C. This resultedin bioactive films.

Example 29

Bioactive materials were obtained by first drop casting the polymer ofexample 14 from THF on glass cover slips (diameter=1.5 cm).Subsequently, three different solutions were made by dissolving: (a) theoligopeptide of example 18; (b) the oligopeptide of example 19; and (c)both oligopeptides of examples 18 and 19 together in THF with 10-30%water. Peptide concentrations of 1, 2, 4 or 8 mole % were used. Thesesolutions were drop cast on the dried polymer film. Most of the times, aslight precipitate was visible. Typically, on one cover slip 1·10⁻⁴ mmolpeptide in the case of 4 mole % peptide and 2.4·10⁻³ mmol polymer wereloaded. The blends on the glass cover slips were dried in vacuo for 2-3days at 35-40° C., resulting in three different oligopeptide-loadedfilms: 29a, 29b, and 29c, that all contained different loadings of theoligopeptides. The samples were sterilized under UV for at least 3hours, prior to use in cell adhesion and spreading experiments or inextraction experiments in vitro.

Example 30

A bioactive material consisting of the polymer of example 14 and theUPy-biotin of example 21 was made via the following method. The polymerof example 8 (0.80 g) was dissolved in THF (2 mL). The white powderobtained in example 21 (34 mg) was added to the THF solution which wassubsequently shaken for a few minutes and spin coated (3500 rpm, 15 s)or drop cast on cleaned glass cover slips (diameter=1.5-2.2 cm). Thesamples were dried for 1 hour in vacuo at room temperature. Thisresulted in UPy-biotin containing bioactive films.

Example 31

A polyethylene glycol with 4H-units was prepared as described in Example9 (12 g), but now after 10 minutes stirring at 140° C., L-ascorbic acid(0.66 g) was added to the polymer melt while stirring the mixture. After5 minutes stirring at 140° C., the polymeric melt was poured in a mouldand cooled down to room temperature. The resulting ascorbic acidcontaining supramolecular polyethylene glycol was obtained as a hardwhite material that slowly released the ascorbic acid upon immersing thematerial in water buffered at pH=7.2 with HEPES (50 mM).

Example 32

Telechelic hydroxy terminated polycaprolacton with a molecular weight of1250 D (3.04 g) and telechelic hydroxy terminated PEO-3000 (5.67 g) wereheated together in vacuo in a 3-neck flask to 120° C. for 30 minutes,followed by the addition of 5 drops dibutyltindilaurate and UPy1 (2.54g). This heterogeneous reaction mixture was subsequently stirred with amechanical stirrer under an argon atmosphere and heated to 150° C. After20 minutes stirring at 150° C., finely grinded 4-acetaminophenol (309mg) was added and stirring was continued for 5 minutes at 140° C. Aftercooling down, a hard semi-flexible white material was obtained thatcontained 2.7% by mass of the bioactive 4-acetaminopheniol. Immersingthe material in water buffered at pH=7.2 with HEPES (50 mM) resulted ina slow release of the bioactive compound.

Examples of Biocompatibility, Biodegradability and Bioactivity ofBioresorbable Supramolecular Material Example 33

Cell culture in vitro: 3T3 mouse fibroblasts were cultured on a 1:1mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium with 10%Fetal Bovine Serum (FBS). They were cultured in a humidified incubatorat 37° C. and 5% CO₂. Before seeding of the cells on the materials, theywere washed twice with PBS solution. Then they were trypsinized with atrypsin-EDTA solution (it was checked with FACS measurements that cellscontain the α₅β₁ integrins irrespective of treatment of the cells withtrypsin-EDTA or with EDTA solution), washed with PBS and counted aftertrypan blue staining in a Neubauer counting chamber. The cells wereseeded in the culture medium (with or without FBS supplemented, asindicated) on the films. The passage of the cells was always between 10and 80 and the viability of the cells was always above 97%.

Cell adhesion and cell spreading experiments: 3T3 mouse fibroblasts(5·10⁴ cells/cm²) were seeded on the cover slips with the supramolecularbioactive materials of example 29 (29a, 29b and 29c) and on cover slipswith the polymer of example 14, on the bottom of a polystyrene culturedish and on glass in 200 μL medium (with or without FBS, as indicated).They were incubated for 5 minutes at room temperature, after which 1 mLmedium (with or without FBS, as indicated) was added. During 1 or 2 daysof culturing in a humidified incubator at 37° C. and 5% CO₂, they werestudied with optical microscopy.

When the mouse 3T3 fibroblasts were cultured on the different bioactiveblends (29a, 29b, 29c; in all cases 4 mole % of peptide was mixed withthe polymer solution prior to preparing the films) and on the polymer ofexample 14 for 2 days in the absence of FBS (Fetal Bovine Serum) toprevent cell adhesion via absorbed serum proteins, aspecific adhesionbut hardly any cell spreading, was already visible after 3 hours on allsamples. However after 1 day, additional cell spreading and the highestdegree of cell adhesion was observed for cells seeded on blend 29c whichmight indicate the possible synergistic effect of the two UPy-peptides.On the film of 29a some cells adhered and spread after 1 day, but lessefficient as on blend 29c. Also less cells spread on mixture 29b after 1day. This is proposed to be due to the fact that PHSRN is a synergisticsequence. These findings for the different films remained unchanged evenafter 2 days, as illustrated in FIG. 1.

Example 34

Inhibition experiments as a comparative study: 3T3 mouse fibroblasts(4-10⁵ cells/mL medium without FBS) were incubated at room temperaturefor 15 minutes with soluble GRGDS peptides (0.3 mM in medium withoutFBS). After this incubation step the cells (6·10⁴ cells/cm²) were seededon the bioactive material 28c (containing 4 mole % of each peptide) in250 μL medium without FBS. In the case of the control the cells (6·10⁴cells/cm²) were not pre-incubated with these soluble GRGDS peptides.After seeding of the cells, they were incubated for 5 minutes at roomtemperature. Then 1 mL medium without FBS was added. After 1 day ofculturing in a humidified incubator at 37° C. and 5% CO₂, they werestudied with optical microscopy which showed that without incubation ofthe cells with soluble GRGDS peptides, the cells adhered and spread outon film 28c after 1 day. However after incubation of the cells with thesoluble GRGDS peptides hardly any adhesion and spreading could bedetected on film 24c after 1 day. This indicates that the cell bindingmight be integrin mediated.

Example 35

Cell binding strength and cell spreading reversibility experiments:Trypsin experiments were performed after 1 day of culturing the 3T3mouse fibroblasts (5·10⁴ cells/cm²) at 37° C. and 5% CO₂ on threedifferent set-ups: on film 28c (containing 4 mole % of each peptide)without FBS added, on film 28c (also containing 4 mole % of eachpeptide) in the presence of FBS, and on the bottom of the polystyreneculture dish in the presence of FBS (PS+FBS). They were all incubated ina trypsin-EDTA solution at room temperature for 30 seconds and 30minutes. After removal of the trypsin-EDTA solution the cells werewashed twice with PBS solution. The cells that remained after thesewashings were incubated again in a humidified incubator at 37° C. and 5%CO₂ for 1 day in cell culture medium without FBS. During the wholeprocess the cells were followed with optical microscopy.

The cells cultured on blend 28c without FBS look similar as cellscultured on blend 28c or on the bottom of a polystyrene (PS) culturedish in the presence of FBS after 1 day of incubation. These resultsindicate that the peptides facilitate cell adhesion and spreading in acomparable manner as the extracellular matrix (ECM) proteins that arepresent in the FBS. Differences, however, can be found in cell bindingstrength experiments using trypsin-EDTA. After 30 seconds of incubationwith trypsin-EDTA, the cells on blend 28c with FBS and on the PS withFBS were completely detached. These cells were subsequently washed offthe plates leaving behind some floating single cells. On the contrary,even after 30 minutes of incubation with trypsin-EDTA the cells on blend28c in the absence of FBS were still adhered and hardly any floatingcells were observed. After removal of the trypsin-EDTA and washing ofthe fibroblasts, they could spread again on blend 28c without FBS whenincubated for 1 additional day, suggesting that the UPy-peptides can actin a reversible fashion. These trypsin experiments indicate that the newsupramolecular materials approach affords strong binding, but that themechanism of binding is sensitive for competitive ECM proteins.

Example 36

In-vivo implantations: Four different solution cast films were prepared:a bioactive film containing 4 mole % of the peptide of example 18 andthe polymer of example 14 (i.e. example 36a), a bioactive filmcontaining both 4 mole % of the peptide of example 18 and 4 mole % ofthe peptide of example 19 and the polymer of example 14 (i.e. example36b), a bare polymer film consisting of the polymer of example 14 (i.e.example 36c) and a bare polymer film consisting of the polymer ofexample 8 (i.e. example 36d). In this case the films were not drop caston cover slips, but on petri dishes. The resulting polymer films had adiameter of 6 mm and were approximately 0.4 mm thick. They were allsubcutaneously implanted in duplicate into male Albino Oxford (AO) rats.The implants with the surrounding tissue were explanted after 2, 5, 10,21 and 42 days of implantation and were embedded in plastic (Technovit7100 cold curing resin based on hydroxyethylmethacrylate (HEMA), KulzerHisto-Technik). The samples were stained with toluidine blue forhistological examination with optical microscopy.

The differences observed after in-vivo implantation between bothsupramolecular materials are striking. At day 5 the cellularinfiltration was very mild for polymers of examples 14 and 8 and a smallfibrous capsule had been formed reflecting the inert and adhesivecharacteristics of the materials. However, in the case of blends 36a and36b vascularization and infiltration of macrophages was observed, whichmight be due to the presence of the peptides that could recruit cellsthrough integrin binding. Another remarkable difference was the factthat in the case of blend 36a and 36b already after 5 days large giantcells were budding into the material from the interface, which indicatesthat the UPy-GRGDS and probably the UPy-PHSRN peptides may not only playa part in the signalling and infiltration of macrophages but also intheir fusion to giant cells.

Giant cells were not detected in the bare polymers and the cellularresponse was negligible up to 42 days. However, the tissue response forboth bioactive blends 36a and 36b became even more active after 10 daysand degradation of the polymer was shown by phagocytotic activity ofgiant cells present in the surrounding tissue. The giant cells at theinterface still did not show any phagocytotic behaviour up to 42 days,although ongoing degradation was observed after 42 days. The resultsafter 21 days of implantation are visible in FIG. 2. The differencesbetween the bioactive blends and the bare polymers are clear. The barepolymers of examples 8 and 14 also behave different. After 42 days thepolymer of example 8 is degraded and could hardly be found back in theanimal, this in contrast with the polymer of example 14 that was hardlydegraded. Also the fibrous capsule in the case of polymer of example 8is much thinner than in the case of polymer of example 14.

Example 37

Degradation studies in vitro: The degradation behaviour of films of thepolymer of example 14 was studied in buffer in the presence of lipaseenzymes, via mass measurements (the dry mass of the samples was measuredon a Sartorius microbalance), differential scanning calorimetry (DSC)and size exclusion chromatography (SEC) after rinsing the samples threetimes with water and drying them at 40° C. for 1.5 hours.

Films of the polymer of example 14 were made via drop casting fromchloroform solution and dried in vacuo at 35-40° C. for 2-3 days priorto use. Samples were shaken in a lipase (from Thermomyces lanuginosus,Aldrich) containing solution which was diluted 1000 times with PBSsolution supplemented with sodiumazide (0.05%) at 37° C. for 23 days.During enzymatic degradation of polymer 14 with the lipase fromThermomyces lanuginosus chain scission was demonstrated with gelpermeation chromatography techniques. After 15 days already 90% massloss was found.

Example 38

The extraction of the peptide of example 18 and the peptide GRGDSwithout a UPy-unit out of films of example 29a was investigated withLC-MS measurements. The amount of peptide that was mixed in thepolymeric materials is in both cases 4 mole %. Calibration was performedby quantification of one fragment of the parent ion (MS²) of thepeptides using different concentrations of the peptides. The surfacearea of the corresponding peak (in the total ion count) was calculatedwith the ICIS algorithm. The extraction experiments were performed asfollows: the film was incubated at 37° C. for 5 minutes in 1 mL water,then the water was removed and the concentration of peptide was measuredwith the described LC-MS procedure (time-point: 5 minutes); another 1 mLwater was added to the film which was subsequently incubated again for 5minutes at 37° C. followed by removal of the water which was analysedwith the LC-MS method (time-point: 10 minutes); another 1 mL water wasadded to the film and the sample was incubated for 10 minutes at 37° C.followed by removal of the water which was analysed with the LC-MSmethod (time-point: 20 minutes); incubation at 37° C. in another 1 mLwater for 20 minutes, followed by removal of the water which wasanalysed with the LC-MS method (time-point: 40 minutes); and incubationat 37° C. in another 1 mL water for 40 minutes, followed by removal ofthe water which was analysed with the LC-MS method (time-point: 80minutes).

These extraction experiments show that dissolution of GRGDS without aUPy-moiety proceeds extremely fast in water at 37° C.; within 5 minutesalmost all of the peptide is dissolved (Table 1). After 80 minutes ofincubation at 37° C. in a total volume of 5 mL water the whole quantityof GRGDS peptide (105%) is dissolved. When a film with 4 mole % GRGDS isincubated for 2 hours at 37° C. in 1 mL water also the whole amount ofGRGDS peptide (101%) is dissolved. The extraction of UPy-GRGDS withwater from the film of example 29a is a slower process. After incubationat 37° C. of film of example 29a containing 4 mole % of UPy-GRGDS for 80minutes in a total volume of 5 mL water ultimately 76% of theUPy-peptide is dissolved (Table 1). However, if this film with 4 mole %UPy-GRGDS is incubated for 2 hours in 1 mL water at 37° C. 64% of theUPy-GRGDS peptide is dissolved. This indicates that the UPy-unit isimportant for the tuneable but dynamic binding of the peptide to thepolymer.

TABLE 1 Extraction experiments on films of polymer of example 14 withUPy-GRGDS (film 29a) or GRGDS: The GRGDS peptide is extracted muchfaster in water than the UPy-GRGDS peptide. extraction extraction timeGRGDS UPy-GRGDS (min) (%) (%) 5 91 39 10 97 60 20 100 76

Example 39

Stability test on bioactive materials m: To test the stability of thebioactive films of examples 29a, 29b and 29c in medium the whole celladhesion and cell spreading experiment was repeated of example 33, butthis time the samples were incubated in medium without FBS (1 mL) in ahumidified incubator at 37° C. and 5% CO₂ for 3 hours, prior to seedingof the cells on the polymers. After this incubation step, the sampleswere washed two times with PBS solution and the cells were cultured inmedium without FBS on these films in a humidified incubator at 37° C.and 5% CO₂ for 1 day. The cells were studied with optical microscopy.This resulted in similar adhesion and spreading patterns as shown before(cf. Figure example 33).

Example 40 Heparin-Containing Supramolecular Hydrogel

Aqueous solutions of acrylamide (1.3 mL; 40% w/v in water) andbisacrylamide (0.6 mL; 2% w/v in water) were mixed. This mixture wasdiluted with tris(hydroxymethyl)aminomethane-buffer (Tris, 1.2 mL; 0.4 MTris-HCl, pH 8.8) and water (1.5 mL) followed by the addition of the4H-unit functionalized heparin of example 25 (123 mg). This mixture washeated to 80° C. and subsequently, UPy6 (48 mg) dissolved in acrylamide(0.20 mL) was added. The mixture was polymerized after the addition ofammoniumpersulfate (50 μL; endconcentration 0.1%) andN,N,N′,N′-tetramethylethylenediamine (TEMED, 2.5 μL; endconcentration0.1%), resulting in a 12% acrylamide gel containing functionalized with4H-units and containing 2% by weight of the bioactive componentaccording to example 24.

Example 41 Uv-Cured Supramolecular Coating

Polymer XI (2.5 g) and UPy6 (1.5 g) were dissolved in hydroxyethylacrylate (HEA, 10 g) together with tetraethyleneglycol diacrylate(TEGDA, 1.0 g), Irgacure 907™ (150 mg, obtained from Ciba, Switzerland)at 80° C. Then a 100 μm film was mechanically drawn on a glass substrateand UV-cured under a nitrogen atmosphere with a Fusion F600 D-bulb (I₀=5W/cm²) with a belt speed of 10.4 m/min, equivalent to a radiation timeof 0.3 s. A clear coating was obtained with good mechanical properties.

1. An implant for biomedical applications having a porous structurecomprising a supramolecular material that comprises: (a) a first polymercomprising at least three 4H-units; and (b) a second polymer that is abioresorbable polymer; wherein the 4H-units have the general formula (3)or formula (4) and tautomers thereof:

wherein X is nitrogen atom or a carbon atom bearing a substituent R⁸ andwherein R¹, R², R³ and R⁸ are independently selected from the groupconsisting of: (i) hydrogen; (ii) C₁-C₂₀ alkyl; (iii) C₆-C₁₂ aryl; (iv)C₇-C₁₂ alkaryl; (v) C₇-C₁₂ alkylaryl; (vi) polyester groups having theformula (5)

wherein R⁴ and Y are independently selected from the group consisting ofhydrogen and C₁-C₆ linear or branched alkyl, n is 1 to 6, and m is 10 to100; (vii) C₁-C₁₀ alkyl groups substituted with 1-4 ureido groupsaccording to the formula (6)R⁵—NH—C(O)—NH—  (6) wherein R⁵ is selected from the group consisting ofhydrogen and C₁-C₆ linear or branched alkyl; and (viii) polyether groupshaving the formula (7)

wherein R⁶, R⁷ and Y are independently selected from the groupconsisting of hydrogen and C₁-C₆ linear or branched alkyl and o is 10 to100; wherein the at least three 4H-units are bonded to the backbone ofthe first polymer via R¹ and R², or via R¹ and R³ with the other Rgroups representing, independently a side chain according to (i)-(viii);wherein the first polymer is derived from polymers having two terminalhydroxyl groups and the first polymer is selected from the groupconsisting of polyesters, polycarbonates, polyorthoesters, polyethers,and copolymers thereof; and wherein the second polymer is different fromthe first polymer and is selected from the group consisting ofpolyethers, polyesters, polyamides, polycarbonates, polyorthoesters,polysaccharides, polyvinylalcohols, and copolymers thereof.
 2. Theimplant according to claim 1, wherein the first polymer is derived fromthe group consisting of polyesters, polycarbonates, and copolymersthereof.
 3. The implant according to claim 1, wherein the first polymeris selected from the group consisting of polycaprolactone, polylactide,polyglycolide, poly(trimethylene) carbonate, poly(1,6-hexanediol)carbonate, and copolymers thereof.
 4. The implant according to claim 1,wherein the second polymer is derived from the group consisting ofpolyesters, polycarbonates, and copolymers thereof.
 5. The implantaccording to claim 1, wherein the second polymer is selected from thegroup consisting of polycaprolactone, polylactide, polyglycolide,poly(trimethylene) carbonate, poly(1,6-hexanediol) carbonate, andcopolymers thereof.
 6. The implant according to claim 1, wherein theporous structure is obtained by a processing method selected from thegroup consisting of freeze drying, particulate leaching,elecro-spinning, spinning, and fused deposition modelling.
 7. Theimplant according to claim 1, wherein the first polymer has a M_(n) of100 to 100,000 and the second polymer has a M_(n) of 100 to 100,000. 8.The implant according to claim 1, wherein the second polymer comprisesone to fifty 4H-units.
 9. A tissue-engineering material comprising theimplant according to claim
 1. 10. A biomedical coating comprising theimplant according to claim
 1. 11. A medical device comprising thebiomedical coating of claim 10, wherein the medical device is selectedfrom the group consisting of stents, catheters, implants, andprostheses.